How it Works
Second Order Non-linear Imaging of Chiral Crystals (SONICC®)
SONICC uses a femtosecond pulsed laser to exploit the frequency-doubling effect found in the majority of protein crystals and produces high-contrast images with negligible background signal.
SONICC has two imaging methods, Second Harmonic Generation (SHG) and Ultraviolet Two-Photon Excited Fluorescence (UV-TPEF). The SHG channel probes crystallinity, and the UV-TPEF channel is specific to proteinaceous samples.
The combined effect of SHG and UV-TPEF imaging is so precise that SONICC can detect microcrystals (<1 um) and distinguish between crystal showers and amorphous aggregates.
Second Harmonic Generation (SHG)
SHG imaging identifies chiral crystals by imaging the sample with infrared light and detecting the frequency-doubled response, only present in chiral crystals. Chiral crystals are those that lack an internal plane of symmetry. Most salt crystals are symmetric and therefore generate no SHG, whereas all protein crystals are chiral and will generate SHG signal.
Nonlinear effects such as SHG require high electric fields, thus requiring the use of a femtosecond (fs) laser. The laser operates with a pulse width of 200 fs and has high peak powers resulting in nonlinear effects, but the pulses are short enough to prevent sample damage associated with localized heating. Further efforts are taken to prevent sample damage by scanning the laser beam quickly so that it does not remain in one spot long enough to heat the sample.
SHG results from all chiral crystals, including some salts and small molecules that form noncentrosymmetric crystals. These chiral salt and small molecule crystals will result in false positives when imaged in SHG mode. To combat this, SONICC is also equipped with Ultraviolet Two-Photon Excited Fluorescence (UV-TPEF) imaging.
Ultraviolet Two-Photon Excited Fluorescence (UV-TPEF)
UV-TPEF imaging is analogous to traditional UV fluorescence and creates images based on the fluorescence of UV excited amino acids such as tryptophan. UV-TPEF is a multi-photon technique, and therefore uses longer wavelengths of excitation versus traditional linear imaging. This provides significant advantages including compatibility with more plates, less damage to protein and confocal imaging.
In order to probe a sample’s fluorescence, the laser is doubled with a nonlinear optical (NLO) crystal from 1064 nm to 532 nm. The green light (532 nm) is then used to image the sample. The two-photon equivalent of the green light is 266 nm, which excites any tryptophan amino acids that are present in the sample. The two-photon excited fluorescence (350 – 400 nm) is then collected and used to create a fluorescence image.
|Fixed Zoom||Compound Zoom||Compound Zoom||Compound Zoom|
|Lens/Objective Options||Asphere 20 mm EFL||Asphere 20 mm EFL||Nikon CFI S Plan Fluor ELWD 20X||40x Nikon CFI S Plan Fluor ELWD 40X|
|Maximum FOL†||2.2 x 2.2 mm||2.2 x 2.2 mm||1.3 x 1.3 mm||0.65 x 0.65 mm|
|Lateral Resolution||4 μm||4 μm||2 μm||1.1 μm|
|Working Distance||13.5 mm||13.5 mm||7.5mm||3.5 mm|
† Continuous zoom is available by controlling the angle of the scanning mirrors.
§ N.A. (numerical aperture) is proportional to the detection limit of SHG.
How Does it Work?
1. A femtosecond fiber laser is used to generate the 1064 nm incident IR light.
2. Depending on the mode selected, the wavelength of the laser used to image the sample is changed from IR to green through a nonliner optical crystal.
3. The sample is raster scanned with a resonant mirror along the fast axis and stepped with a galvo-driven mirror for the slow scan axis.
4. A high numerical aperture (NA) objective is used to focus the laser beam onto the sample.
5. The green SHG signal is collected in the transmission direction.
6. UV fluorescence generated in the UV-TPEF mode is collected in the reflected (epi) direction.
7. Photo Multiplier Tubes (PMTs) detect the nonlinear response via photon counting which images are created from.